Biochemical data indicate that eukaryotic Kir channels form hetero- or homo-tetramers [5, 19]. Kir2.1 channels readily form homo-tetramers, as well as hetero-tetramers with other Kir2 family members such as Kir2.2 and Kir2.3 [5, 20]. One of the hallmarks of Kir2 channels is strong inward rectification, in which K+ ions flow preferentially into rather than out of the cell [8]. Inward rectification is produced by cytosolic polyamines and Mg2+ occluding the ion conductance pathway as K+ ions are flowing outward. These positively charged particles are then removed from the pore when K+ ions flow into the cell [9]. Rectification is the primary means of gating for Kir2 channels [10-12]. The rate of rectification by these positively charged polyamine and Mg2+ block varies between different members of the Kir family depending on the presence of key hydrophilic residues located at two major locations. The strongest binding site of these blockers resides within the trans-membrane site defined by Asp172 in Kir2.1 and weaker, recruiting sites are comprised of several positive and negative charged residues lining the pore in the cytoplasmic domain [9, 13, 14]. The overall effect of rectification allows Kir2.1 to play a key role in eukaryotic cells by driving the resting membrane potential to EK when the cell is at rest [8].
In addition to rectification, Kir channels possess other gating elements. For example, Kir2.1 channels are opened by the binding of the membrane phospholipid PIP2 directly to the channel [15]. Kir channels also can interact with certain alcohols [29, 30, 31]. Using the functional definition of gates as energetic barriers that block the permeation of potassium, it was proposed that a flexible loop, named the G-loop, at the junction between the cytoplasmic and transmembrane domains, can occlude the permeation pathway as an additional gating element [14, 16]. Therefore, at least two energetic barriers, one located inside the pore-lining M2 helix and the other formed by the G-loop, may operate to control the ionic flow. Of the two, the G-loop is unique by being located in the cytoplasmic domains and is too narrow to permit permeation in the absence of PIP2 [14]. Unlike the T1 domain of voltage-gated K channels for which ions flow through side openings created between T1 and transmembrane domains [17], K ions flow through the central pore of Kir2.1 channels beginning at the cytoplasmic domain and continuing to the selectivity filter. How the G-loop and M2 helix regions work together to control ionic flow is unknown. Studies with chimeric KcsA/Kir2.1 channels indicate the cytoplasmic domain is required for Kir2.1 gating [18].